Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique
Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique
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DOI：
10.1016/j.chroma.2007.05.054
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发表时间：
2007-08-03
影响因子：
4.1
通讯作者：
Tomita, Masaru
中科院分区：
文献类型：
--
作者：
Soga, Tomoyoshi;Ishikawa, Takamasa;Tomita, Masaru
建立了一种基于压力辅助毛细管电泳电喷雾电离质谱仪(PACE-MS)测定核苷酸的方法。为了防止多个磷酸化物种吸附到熔融石英毛细管上，通过用含有磷酸盐的背景电解液对毛细管进行预处理，硅醇基被磷酸根离子掩盖。在预处理过程中，关闭雾化气体，以避免磷酸根离子污染MS检测器。要在pH 7.5的条件下使用CE正模式检测核苷酸，需要在电泳过程中对进样毛细管施加气压，以补充朝向阴极的电渗流(EOF)。此外，我们还使用缓冲液补充系统交换了每次分析的运行电解液，以获得所需的重复性。在优化的条件下。在不到20min的时间内，对核苷酸、烟酰胺-腺嘌呤二核苷酸和辅酶A(CoA)化合物等14个磷酸化空间进行了很好的测定。方法的相对标准偏差(n=6)对迁移时间优于0.9%，对峰面积的相对标准偏差在1.7%~8.1%之间。在信噪比为3的条件下，以50mbar的压力进样30nL的S(30 NL)，对这些物种的检出限在0.5~1.7Mol/L之间。与以前的方法相比，该方法具有可靠和定量的特点，并通过对大肠杆菌野生型、pfkA和pfkB基因敲除突变株细胞内核苷酸和辅酶A化合物的分析证明了该方法的有效性。该方法被用来表明pfkA是主要的功能酶。(C)2007 Elsevier B.V.保留所有权利。
A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions. 14 phosphorylated spacies such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1 % for peak areas. The detection limits for these species were between 0.5 and 1.7 mu mol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme. (c) 2007 Elsevier B.V. All rights reserved.